Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines.

Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

Evaluation of the protective effect of immunization spf DBA/2J mice with selected bacterial, recombinant Hsp60 antigens during Salmonella Enteritidis challenge.

Salmonella Enteritidis is one of the most common causes of food poisoning in humans. Many attempts have been made to develop an effective vaccine against S. Enteritidis for use in poultry, but experiments aimed at the complete elimination of this pathogen from poultry farms have not provided satisfactory results. The development of new generation vaccines against salmonellosis, such as subunit vaccines based on heat shock proteins (HSPs), is strongly justified. The high immunogenicity of Hsp60 isolated from Procaryota, including Salmonella, has been suggested by the presence of IgG anti-Hsp60 antibodies in mice immunized with these proteins. The aim of the studies was to evaluate the protective effects of immunization with recombinant Hsp60 from selected gram-negative bacteria (S. Enteritidis, Escherichia coli, Pasteurella multocida, Histophilus somni) in spf DBA/2 J mice experimentally infected with S. Enteritidis. The study demonstrated that double subcutaneous immunization of mice with a dose of 10 µg rHsp60 induced a specific immune response of IgG antibodies in tested animals. The median lethal dose (LD50) for the murine model spf DBA/2 J orally infected with S. Enteritidis was estimated at 6.84 × 105 cfu/animal. Mice immunized with rHsp60 from gastrointestinal pathogens (S. Enteritidis and E. coli) showed better survival after experimental infection with a 3 × LD50 dose from S. Enteritidis, compared to animals immunized with proteins obtained from respiratory pathogens (P. multocida and H. somni). However, the log-rank analysis did not show significant differences in the survival rates between rHsp60-immunized mice and controls. S. Enteritidis was not isolated any less frequently from internal organs and faeces of rHsp60-immunized mice than from controls. Nevertheless, the level of haptoglobin (but not IL-6) was increased in all mice in which the presence of the pathogen was observed. Bacterial Hsp60 is an interesting candidate for a subunit vaccine, but its use in livestock animals must be further investigated.

Comparative evaluation of Salmonella Typhimurium vaccines derived from UK-1 and 14028S: Importance of inherent virulence.

The initial virulence and invasiveness of a bacterial strain may play an important role in leading to a maximally efficacious attenuated live vaccine. Here we show that χ9909, derived from Salmonella Typhimurium UK-1 χ3761 (the most virulent S. Typhimurium strain known to us), is effective in protecting mice against lethal UK-1 and 14028S (less virulent S. Typhimurium strain) challenge. As opposed to this, 14028S-derived vaccine χ12359 induces suboptimal levels of protection, with survival percentages that are significantly lower when challenged with lethal UK-1 challenge doses. T-cell assays have revealed that significantly greater levels of Th1 cytokines IFN-γ and TNF-α were secreted by stimulated T-lymphocytes obtained from UK-1(ΔaroA) immunized mice than those from mice immunized with 14028S(ΔaroA). In addition, UK-1(ΔaroA) showed markedly higher colonizing ability in the spleen, liver, and cecum when compared to 14028S(ΔaroA). Enumeration of bacteria in fecal pellets has also revealed that UK-1(ΔaroA) can persist in the host for over 10 days whereas 14028S(ΔaroA) titers dropped significantly by day 10. Moreover, co-infection of parent strains UK-1 and 14028S resulted in considerably greater recovery of the former in multiple mucosal and gut associated lymphatic tissues. Mice immunized with UK-1(ΔaroA) were also able to clear UK-1 infection remarkably more efficiently from the target organs than 14028S(ΔaroA). Together, these results provide ample evidence to support the hypothesis that attenuated derivatives of parent strains with higher initial virulence make better vaccines.

Incorporation of membrane-anchored flagellin into Salmonella Gallinarum bacterial ghosts induces early immune responses and protection against fowl typhoid in young layer chickens.

The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of Salmonella Gallinarum can enhance protective immune responses against fowl typhoid, a septicemic disease of poultry, in chickens. BGs are empty cell envelopes derived from Gram-negative bacteria through the bacteriophage phiX174 gene E mediated lysis. In this study, the S. Gallinarum ghosts carrying flagellin were genetically constructed utilizing a lysis plasmid pJHL184-flagellin, designed for the coexpression of the flagellin and the lysis protein E. The adjuvant effect of flagellin was evaluated by immunizing seven day old brown nick layer chicks once orally with either S. Gallinarum-flagellin (SG-fliC) ghosts or S. Gallinarum (SG) ghosts alone. Our results showed that immunization with the SG-fliC ghosts elicited early and higher systemic (IgG) and mucosal (IgA) antibody responses compared to the SG ghosts alone, although not always statistically significant. Flow cytometric analysis of the CD3 + CD4+ and the CD3 + CD8+ T cell populations in peripheral blood mononuclear cells were higher in chickens immunized with the SG-fliC ghosts compared to the chickens vaccinated with the SG ghosts alone. Furthermore, the chickens immunized with SG-fliC ghosts exhibited significantly (p < 0.05) higher IL-6 and IFN-γ responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent S. Gallinarum wild type strain at 28th day post immunization, 5 of 10 birds died (50%) in case of SG-fliC ghost group while 60% (6 of 10 birds died) mortality was observed in the SG ghost group. Collectively, these results suggest that the expression of flagellin in SG ghosts improves antigen-specific humoral and cell mediated immune responses, and can enhance protective efficacy of the BG-based vaccines against the virulent challenges.

Immune response of turkey poults exposed at 1 day of age to either attenuated or wild Salmonella strains.

Salmonellosis is a foodborne zoonosis that is most often acquired by consuming poultry products such as eggs and poultry meat. Amongst other measures the vaccination of food-producing poultry is thought to contribute to a reduction in human salmonellosis. In the European Union (EU) in 2014 the licence of a commercially available Salmonella vaccine for chickens and ducks was extended to turkeys. In the present study, we examined the course of infection with a virulent Salmonella enterica ssp. enterica serovar Enteritidis (SE) strain, a virulent S. enterica ssp. enterica serovar Typhimurium (ST) strain, and the respective live vaccine containing attenuated strains of both serovars in turkey poults. Besides collecting microbiological data and detecting invading Salmonella in the caecal mucosa via immunohistochemistry, we also assessed immune reactions in terms of antibody production, influx of CD4-, CD8α- and CD28-positive cells into the caecal mucosa and the expression of four different immune-related proteins. We found that the attenuated strains were able to invade the caecum, but to a lower degree and for a shorter duration of time compared to virulent strains. Infections with virulent Salmonellae also caused an increase in CD4-, CD8α- and CD28-positive cells in the caecal mucosa and an increased transcription of iNOS, IL-8-like chemokines, and IFN-γ. In poults treated with attenuated bacteria we could not detect any evidence of immune responses. In conclusion, the vaccine showed a lower degree of caecal invasion and induced weaker immune reactions compared to the virulent Salmonella strains in turkeys. The efficiency of the vaccine has to be verified in future studies.

A Live Salmonella Gallinarum Vaccine Candidate Secreting an Adjuvant Protein Confers Enhanced Safety and Protection Against Fowl Typhoid.

Live attenuated vaccines are used for effective protection against fowl typhoid (FT) in domestic poultry. In this study, a lon/cpxR/asd deletion mutant of Salmonella Gallinarum expressing the B subunit of a heat labile toxin (LTB) from Escherichia coli, a known adjuvant, was cloned in a recombinant p15A ori plasmid, JOL1355, and evaluated as a vaccine candidate in chickens. The plasmid was shown to be stable inside the attenuated Salmonella Gallinarum cell after three successive generations. Moreover, from an environmental safety point of view, apart from day 1 the JOL1355 strain was not detected in feces through day 21 postinoculation. For the efficacy of JOL1355, a total of 100 chickens were equally divided into two groups. Group A (control) chickens were intramuscularly inoculated with phosphate-buffered saline at 4 and 8 wk of age. Group B chickens were primed and boosted via the intramuscular route with 200 µL of a bacterial suspension of JOL1355 containing 1 × 10(8) colony forming units. All the chickens in Group A and B were challenged at 3 wk postbooster by oral inoculation with a wild-type Salmonella Gallinarum strain, JOL420. The JOL1355-immunized group showed significant protection and survival against the virulent challenge compared to the nonimmunized group. In addition, Group B exhibited a significantly higher humoral immune response, and the chickens remained healthy without any symptoms of anorexia, diarrhea, or depression. Group B also exhibited a significantly lower mortality rate of 4% compared to the 46% of the control group, which can be attributed to higher immunogenicity and better protection. The Group B chickens had significantly lower lesion scores for affected organs, such as the liver and spleen, compared to those of the control chickens (P < 0.01). These findings suggest that JOL1355 is a promising candidate for a safe and highly immunogenic vaccine against FT.

Improving adjuvant systems for polyclonal egg yolk antibody (IgY) production in laying hens in terms of productivity and animal welfare.

The antibody production in the egg yolks of immunized laying hens is seen as a way of improving animal welfare compared with conventional production by mammals. Immunoglobulin Y (IgY) technology, however, has still to address welfare issues linked to the widespread use of an adjuvant in vaccines. Currently, Freund's adjuvants, complete (FCA) or incomplete (FIA), remain the standard. This study sought to evaluate various approaches used to enhance egg yolk antibody production in terms of both productivity and avian welfare. The outer membrane protein (OMP) of Salmonella Typhimurium was used as the prototype antigen. At 20 weeks of age, 56 ISA Brown hens, with specific-Salmonella-free status, were divided into seven groups (n=8) and received an initial intramuscular immunization. Hens in the two negative control groups received phosphate buffered saline (PBS) or FIA alone. Hens in the other groups received 80µg of Salmonella OMP emulsified with one of the following adjuvants: 200µl of FIA alone (T1); 200µl of FIA supplemented with 8µg of C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) (T2); and 280µl of Montanide ISA 70 VG (T4). Birds in the T3 group received the antigen in emulsion with FIA and were given the tested immunostimulatory component (l-carnitine) via their feed (100mg/kg). A positive control group (PC) received FCA for the first and final immunizations and FIA for the other boosters. Immunization was repeated after 20, 46, 82 and 221 days. Eggs were collected regularly until 242 days after the first immunization and the anti-Salmonella Typhimurium activities in the yolk were determined by ELISA. After 242 days, the birds were euthanized and the injection sites were evaluated for gross and microscopic lesions. Among the tested immunostimulatory approaches, supplementation of FIA with CpG-ODN led to a significant and long-lasting enhancement of the specific antibody response. This treatment was even higher than the positive benchmark using FCA in the first immunization. The study results showed that a clinical examination of injection sites is insufficient for drawing conclusions about the local tolerance of vaccines. Tissue damage was noticeable in all treatment groups. The birds receiving the Montanide adjuvant, however, had fewer and less severe lesions. Given these limited side-effects, Montanide ISA 70 VG could provide the depot effect needed to ensure the immunomodulatory efficiency of CpG-ODN. The association of these two adjuvants could prove a promising alternative to Freund's adjuvants (FA).

Application of pH-sensitive fusogenic polymer-modified liposomes for development of mucosal vaccines.

To evaluate the usefulness of pH-sensitive fusogenic polymer (succinylated poly(glycidol) (SucPG) and 3-methylglutarylated poly(glycidol) (MGluPG))-modified liposomes as mucosal vaccine in the induction of a protective immune responses was evaluated. Mice were nasally immunized with OVA-containing SucPG-modified liposomes. After immunization, significant Ag-specific Abs were detected in the serum and intestine. When sera were analyzed for isotype distribution, antigen-specific IgG1 Ab responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ and IL-4 mRNA were detected. The same result was obtained also in the mouse immunized with OVA-containing MGluPG-modified liposomes. Furthermore, we examined the induction of immune responses in chickens following intraocular immunization with Salmonella Enteritidis Ag-containing MGluPG-modified liposomes, and the protective effect against the challenge with S. Enteritidis. Immunization with S. Enteritidis Ag-containing MGluPG-modified liposomes induced significant Ab responses against S. Enteritidis in the serum and intestine. Less fecal excretion of bacteria was observed in chickens immunized with S. Enteritidis Ag-containing MGluPG-modified liposomes after challenge. The numbers of bacteria in the caecum were also lower in immunized chickens than in unimmunized controls.

Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice.

OBJECTIVES: Typhoid fever is caused by Salmonella enterica serovar Typhi. OmpC, OmpF and OmpA, the three major outer membrane proteins (OMPs), could serve as vaccine candidates. METHODS: The porins antigenicity was predicted in silico. The OMP genes were amplified, cloned and expressed. Sero-reactivities of the recombinant proteins purified by denaturing method were assayed by ELISA. BALB/c mice were immunized with the recombinant porins followed by bacterial challenge. RESULTS: Bacterial challenge of the animal model brought about antibody triggering efficacy of the antigen in OmpF > OmpC > OmpA order. Experimental findings validated the in silico results. None of the antigens had synergic or antagonistic effects on each other from immune system induction points of view. Despite their high immunogenicity, none of the antigens was protective. However, administration of two or three antigens simultaneously resulted in retardation of lethal effect. Porins, in addition to their specific functions, share common functions. Hence, they can compensate for each other's functions. CONCLUSIONS: The produced antibodies could not eliminate the pathogenicity by blockade of one or some of the antigens. Porin antigens are not suitable vaccine candidates alone or in denatured forms. Native forms of the antigens maybe studied for protective immunogenicity.

CpG oligonucleotides and recombinant interferon-γ in combination improve protection in chickens to Salmonella enterica serovar Enteritidis challenge as an adjuvant component, but have no effect in reducing Salmonella carriage in infected chickens.

Salmonella enterica serovar Enteritidis is the most common cause of human salmonellosis in many developed nations. It is frequently associated with both poultry meat and eggs. In the present study we have determined whether CpG oligonucleotides that stimulate the immune system via Toll like-receptors 15 and 21 in the chicken can be used as immunomodulatory agents to break carriage of S. Enteritidis in in vitro and in vivo infection models. We also investigated its use as a component in an adjuvant to stimulate cell mediated immunity with a killed vaccine preparation. Following infection of the chicken macrophage-like cell line HD11 with Salmonella enterica serovar Gallinarum, cells were stimulated with an oligonucleotide containing a CpG motif, or with a non-CpG oligonucleotide control at concentrations ranging from 0 to 80 µM. Addition of the CpG oligonucleotide greatly enhanced clearance of S. Enteritidis in dose-dependent manner, whilst the control oligonucleotide had no significant effect. In contrast, stimulation of cells infected with S. Gallinarum had no effect. The CpG or control oligonucleotide with recombinant chicken interferon-γ was administered intramuscularly into chickens experimentally colonized with S. Enteritidis, although neither preparation produced any change in intestinal colonization levels to that in untreated control birds. Finally, CpG oligonucleotides were incorporated with recombinant interferon-γ, double-stranded RNA (Poly I:C) and squalene as a Th1-stimulating combined adjuvant for synergistic activation of cellular immunity (CASAC) together with whole killed Salmonella as the antigen as an experimental vaccine. Following vaccination and challenge of chickens with S. Enteritidis, CASAC gave significant protection to intestinal colonization whereas the same antigen given with a proprietary adjuvant did not. Neither adjuvant increased protection to systemic infection. The data suggest that adjuvants incorporating CpG motifs and interferon-γ may improve protection afforded by killed-Salmonella vaccines.

Suppression of murine melanoma growth by a vaccine of attenuated Salmonella carrying heat shock protein 70 and Herpes simplex virus-thymidine kinase genes.

Attenuated Salmonella can invade tumor cells and acts as a eukaryotic expression vector for gene propagation. We constructed a bi-gene, eukaryotic co-expression DNA vaccine of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and Herpes simplex virus-thymidine kinase (HSV-tk) and used attenuated Salmonella as a vector to treat murine melanoma. In vitro, recombinant Salmonella can carry plasmid stably and can invade into the cytoplasm of B16 tumor cells expressing the protein of the mtHSP70/HSV-tk gene by Western blot assay. In vivo, after the recombinant Salmonella was injected into tumors, the HSV-tk precursor drug ganciclovir (GCV) was administered to start the HSV-tk killing of tumor cells. We found that the mtHSP70/HSV-tk recombinant bacteria can raise CD8+ T lymphocytes in peripheral blood by flow cytometry and in tumor tissues by immunofluorescence detection, increase IFN­Î³ contents in tumor tissue by ELISA and significantly suppress tumor growth.

Evaluation of recombinant Salmonella expressing the flagellar protein fliC for persistence and enhanced antibody response in commercial turkeys.

Salmonella enterica serovar Enteritidis (SE) is one of the most common causes of human foodborne illness in the United States. Previous research indicates that antibodies against the fliC protein can provide protection against Salmonella challenge in mice. To generate a vaccine that effectively protects poultry against multiple Salmonella serotypes, novel attenuated strains of SE were developed to express a fliC peptide sequence on the outer membrane protein lamB in association with an M2e (marker) epitope. In 3 separate trials, poults were immunized with 10(7) to 10(8) cfu/poult of the appropriate recombinant Salmonella strains (ΔSE-M2e or ΔSE-M2e-fliC) via oral gavage on the day of hatch and again on d 21 posthatch. Liver, spleen, and cecal tonsils were aseptically removed on d 7, 14, 21, 28, 35, and 42 posthatch for detection of Salmonella, and blood samples were obtained at these same time points for determination of an M2e-specific antibody response. In all 3 trials, the ΔSE-M2e-fliC strain exhibited significantly less invasion of the liver and spleen at d 7 and 14 when compared with ΔSE-M2e or SE phage type 13A (P < 0.05). Similarly, colonization of the cecal tonsils was decreased in the poults immunized with the ΔSE-M2e-fliC strain. By d 21, the ΔSE-M2e-fliC strain exhibited a significantly higher M2e-specific antibody response when compared with the negative control and SE phage type 13A groups (P < 0.05). However, no significant differences in M2e-specific antibody responses were observed between the ΔSE candidate vaccine strains throughout the study. Overall, these data suggest that oral live attenuated Salmonella-vectored vaccines expressing a fliC peptide sequence are able to elicit a humoral immune response in commercial poults and may contribute to a reduction in Salmonella organ invasion and colonization.

Recombinant attenuated Salmonella harboring 4-1BB ligand gene enhances cellular immunity.

OBJECTIVE: To transfect antigen presenting cells (APCs) with 4-1BB ligand DNA by attenuated Salmonella enterica serovar Typhimurium in vivo, and to observe the effects of ectogenous 4-1BBL on the immune functions of infected rats. METHODS: Attenuated Salmonella typhimurium (vaccine strain) carrying plasmids pIRES2-EGFP-4-1BBL was constructed and used to infect HepG2 hepatoma cells. The expression of reporter gene, green fluorescent protein (GFP) and rat 4-1BBL in the transfected cells was detected by double-immunofluorescence staining. Rats were fed with the recombinant bacteria intragastrically on three occasions in 2 weeks, and were then sacrificed. The transcription and expression of GFP and 4-1BBL genes in splenocytes were measured by RT-PCR and flow cytometry. The phenotypes of T cells in peripheral blood and splenocytes were determined by flow cytometry. The content of IFN-gamma in the cultural supernatant of splenocytes stimulated by PHA was measured by ELISA. RESULTS: The recombinant bacteria harboring 4-1BBL had the same invasive abilities as the original bacteria, and it was able to deliver exogenous genes into HepG2 cells, where the GFP and 4-1BBL were successfully expressed. There were significant upregulations of CD3(+)CD8(+) T cells (P=0.018) and CD3(+)CD25(+) T cells (P=0.019) in the peripheral blood cells as well as CD3(+)CD8(+) T cells (P=0.022), and CD3(+)CD25(+) T cells (P=0.008) in splenocytes of the infected rats. The rats had more 4-1BBL expression detected in the spleen. IFN-gamma released by PHA-stimulated splenocytes increased significantly by the recombinant bacteria as compared with controls (P=0.002). CONCLUSION: Salmonella serovar Typhimurium containing 4-1BBL can transfect target genes into antigen presenting cells in vivo, and the expression of exogenous 4-1BBL enhances cellular immunity markedly.

Evaluation of new generation Salmonella enterica serovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA.

Increasing the immunogenicity to delivered antigens by recombinant attenuated Salmonella vaccines (RASV) has been the subject of intensive study. With this goal in mind, we have designed and constructed a new generation of RASV that exhibit regulated delayed attenuation. These vaccine strains are phenotypically wild type at the time of immunization and become attenuated after colonization of host tissues. The vaccine strains are grown under conditions that allow expression of genes required for optimal invasion and colonization of host tissues. Once established in the host, these virulence genes are turned off, fully attenuating the vaccine strain. In this study, we compared 2 of our newly developed regulated delayed attenuation Salmonella enterica serovar Typhimurium strains chi9088 and chi9558 with the Deltacya Deltacrp Deltaasd strain chi8133, for their abilities to express and present a secreted form of the alpha-helical region of pneumococcal surface protein A (PspA) to the mouse immune system. All 3 strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens in orally immunized mice. However, both RASVs expressing delayed attenuation elicited significantly greater anti-PspA immune responses, including serum IgG and T cell secretion of IL-4 and IFN-gamma, than other groups. Also, vaccination with delayed attenuation strains resulted in a greater degree of protection against Streptococcus pneumoniae challenge than in mice vaccinated with chi8133 (71-86% vs. 21% survival, P

Effects of a commercially available vaccine against Salmonella enterica serotype Newport on milk production, somatic cell count, and shedding of Salmonella organisms in female dairy cattle with no clinical signs of salmonellosis.

OBJECTIVE: To determine effects of vaccination with siderophore receptor and porin (SRP) proteins derived from Salmonella enterica serotype Newport on milk production, somatic cell count, and shedding of Salmonella organisms in female dairy cattle. ANIMALS: 180 female Holsteins. PROCEDURES: Cattle were randomly assigned to receive Salmonella Newport SRP vaccine or control solution. Vaccine or control solution was injected 45 to 60 days before parturition, and cattle received a second dose 14 to 21 days before parturition. Milk production was monitored for the first 90 days of lactation. Feces for isolation of Salmonella and blood samples for detection of antibodies against Salmonella Newport were collected at day of first injection and at days 7 to 14 and 28 to 35 of lactation. RESULTS: Cattle inoculated with Salmonella Newport vaccine produced significantly more milk (1.14 kg/d), compared with cattle injected with the control solution. Cattle administered the vaccine had significantly higher concentrations of circulating antibody against Salmonella Newport SRP proteins at 7 to 14 days and 28 to 35 days of lactation. Salmonella Newport was not recovered; however, Salmonella enterica serotype Agona was recovered from 31 (20.3%) cattle, but likelihood of recovery did not differ significantly between vaccinates and control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of a vaccine against Salmonella Newport SRP proteins to healthy dairy cattle prior to parturition increased milk production, even in cattle without detectable shedding of Salmonella Newport or clinical signs of salmonellosis. Additional research is needed to clarify the mechanisms by which productivity was improved.

Protective effect of atorvastatin on acute systemic inflammation-induced endothelial dysfunction in hypercholesterolaemic subjects.

AIMS: Recent studies suggest an association between acute inflammation and deterioration of arterial function. The effect of acute inflammation on endothelial function and the role of treatment with statins have not been investigated in subjects with dyslipidaemia. METHODS AND RESULTS: In this randomized, placebo-controlled, double-blind study, we generated a transient systemic inflammation by Salmonella typhi vaccination in 50 volunteers with mild hypercholesterolaemia after 4 days of treatment with atorvastatin 40 mg or placebo once daily. Endothelium-dependent flow-mediated dilation (FMD) of the brachial artery and circulating levels of endothelial and inflammatory markers were measured before and 8 h after the vaccine. Vaccination produced a decline on FMD at 8 h (absolute decrease of 2.55%, P = 0.001), indicating an unfavourable effect on endothelial function. In contrast, in atorvastatin-treated subjects, FMD was preserved after vaccination (decrease of 0.15%, P = 0.005 vs. placebo). The vaccination-induced decline in plasma level of nitric oxide metabolites (by 6.0 micromol/L, P = 0.007) and antioxidant capacity (by 20.6 micromol/L, P = 0.001) in the placebo group were completely abolished by atorvastatin (P = 0.038 and P = 0.005, respectively, vs. placebo). In contrast, atorvastatin had no significant effect on cytokine levels. CONCLUSION: Acute inflammation is aetiologically associated with the deterioration of vasomotor and systemic endothelial function in hypercholesterolaemic patients. Atorvastatin effectively abrogates these deleterious effects.

Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis.

Regulatory T (T(reg)) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on autoantigen-reactive T(reg) cells, stimulation to myelin-independent Ags may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain T(reg) cell dependency, a kinetic analysis was performed showing increased levels of FoxP3(+)CD25(+)CD4(+) T cells. Inactivation of these T(reg) cells resulted in loss of protection. Adoptive transfer of the vaccine-induced T(reg) cells protected mice against EAE with greater potency than naive or Salmonella vector-induced T(reg) cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these T(reg) cells in conjunction with immune deviation by Th2 cells optimally induced protective T(reg) cells when compared those induced in the absence of Th2 cells. These data show that T(reg) cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.

Cancer immunotherapy based on killing of Salmonella-infected tumor cells.

A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella-specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.

[Specific anti-glioma angiogenesis immune response induced by attenuated Salmonella typhimurium vaccine expressing vascular endothelial growth factor receptor-2].

BACKGROUND & OBJECTIVE: Vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2) play important roles in tumor angiogenesis. This study was to investigate anti-vasculature and anti-glioma effects of attenuated Salmonella typhimurium vaccine expressing VEGFR2 gene. METHODS: Plasmid pcDNA3.1-VEGFR2 was constructed, and electrotransfected into attenuated Salmonella typhimurium strain SL7207. C57BL/6J mice were immunized with gastrogavage of cDNA vaccine encoding VEGFR2 (vaccine group). C57BL/6J mice received gastrogavage of pcDNA3.1 or NaHCO3 were used as controls. Serum level of specific anti-VEGFR2-IgG antibody was detected by ELISA. Cytotoxic T lymphocytes (CTLs) activity was measured by MTT assay. Mouse models of intracranial Gl261 glioblastoma were treated with gastrogavage of attenuated Salmonella typhimurium expressing VEGFR2 gene. Tumor diameter was measuredu microvessel density (MVD) was detected by immunohistochemistryu tumor cell apoptosis was detected by TUNEL. RESULTS: All mice immunized with the vaccine developed high levels of anti-VEGFR2-IgG antibody, and showed strong CTLs activities against VEGFR2. The vaccine substantially inhibited glioblastoma growth. MVD was significantly lower in vaccine group than in pcDNA3.1 group, and NaHCO3 group (8.8+/-1.9 vs. 27.2+/-4.5, and 26.5+/-5.8, P < 0.01)u while apoptotic cell count per visual field was significantly higher in vaccine group than in the rest 2 groups (23.4+/-4.7 vs. 3.1+/-1.0, and 4.4+/-1.2, P < 0.01). CONCLUSION: Attenuated Salmonella typhimurium vaccine expressing VEGFR2 gene can break immunologic tolerance against self-VEGFR2 antigen, and specifically kill glioblastoma vascular endothelial cells by inducing specific anti-VEGFR2 immunoreaction.

Anti-angiogenesis effect on glioma of attenuated Salmonella typhimurium vaccine strain with flk-1 gene.

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.